This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding glucose-6-phosphate/phosphate translocators in plants and seeds.
During C3 photosynthesis, energy from solar radiation is used for the formation of phosphorylated C3 sugar phosphates, triose phosphates (triosP), and 3-phosphoglycerate (3-PGA). These products are then exported from the chloroplasts into the cytosol via the trioseP/3-PGA/phosphate translocator (TPT). In the mature leaves of most plants, the exported photosynthates are then used in the formation of sucrose, which is allocated via the phloem to the heterotrophic plant organs, such as young leaves, roots, seeds, fruits or tubers. In these sink tissues, sucrose serves as a source of carbon and energy and is first cleaved by the action of invertases or sucrose synthase. The products of these reactions are converted into hexose phosphates.
Nongreen plastids of heterotrophic tissues import carbohydrates to fuel metabolism in those cells. Amyloplasts, found in storage tissues, convert the carbohydrates into starch which is efficiently stored. In general, the plastids of heterotrophic tissues cannot generate hexose phosphates from C3 compounds due to an absence of fructose 1,6-bisphosphatase activity. Thus, the TPTs that transported the triose-phosphates out of the chloroplasts in photosynthetic tissues are not useful for transporting them back into the storage plastids of heterotrophic tissues. Therefore, nongreen plastids rely on the import of hexose phosphates to supply the materials for starch biosynthesis, and for generating energy through the oxidative pentose phosphate pathway. A hexose-phosphate/phosphate translocator (or transporter, or antiporter, which are used interchangeably herein) allows membrane passage of hexose-phosphate while simultaneously transporting inorganic phosphate, or triose-phosphates, in the opposite direction. In nongreen plant tissues glucose 6-phosphate (Glc6P) is the preferred hexose phosphate taken up by nonphotosynthetic plastids. The translocation event is selective, as shown by the inability of amyloplast membranes to transport phosphoenolpyruvate, fructose-6-phosphate, or glucose-1-phosphate. It has been shown recently (Kammerer, B. et al. (1998) The Plant Cell 10:105-117) that Glc6P is taken up by nongreen plastids via a glucose-6-phosphate/phosphate translocator (GP/PT). Glucose-6-phosphate imported by the GP/PT protein can be incorporated into starch, releasing inorganic phosphate, or can serve as a substrate for the oxidative pentose phosphate pathway, yielding triose phosphates.
GP/PT is one of the main translocators to provide plastids with carbon for biosynthetic pathways and energy. GP/PT transcripts are abundant in heterotrophic tissues active in starch synthesis, such as potato tubers, maize kernels, and pea roots (Kammerer et al. (1998) Plant Cell 10:105-117), but are barely detectable in photosynthetic tissues. However, endosperm-specific starch synthesis, found in maize and barley, occurs in the cytosol and does not utilize GP/PT transport (Denyer et al. (1996) Plant Physiol 112:779-185; Thorbjornsen et al. (1996) Plant J 10:243-250). So not all starch containing tissues are necessarily dependent upon GP/PT activities. Still, it is clear that GP/PTs play an integral role in maintaining starch synthesis and energy metabolism in normal plant growth and development. Accordingly, the availability of nucleic acid sequences encoding all or a portion of the GP/PT protein would facilitate studies to better understand hexose phosphate transport, provide genetic tools for the manipulation of biosynthetic pathways and energy production, and may provide possible targets for herbicides or the engineering of herbicide resistance.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 401 amino acids that has at least 70% identity based on the Clustal method of alignment when compared to a rice glucose-6-phosphate/phosphate translocator (GP/PT) polypeptide of SEQ ID NO:2, a nucleotide sequence encoding a first polypeptide of at least 395 amino acids that has at least 86% identity based on the Clustal method of alignment when compared to a soybean GP/PT polypeptide of SEQ ID NO:4, and a nucleotide sequence encoding a first polypeptide of at least 302 amino acids that has at least 93% identity based on the Clustal method of alignment when compared to a wheat GP/PT polypeptide of SEQ ID NO:6. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, and 5, that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, and 6. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 60 (preferably at least one of 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, and 5, and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a GP/PT polypeptide of at least 401 amino acids that has at least 70% identity based on the Clustal method of alignment when compared to a glucose-6-phosphate/phosphate translocator polypeptide of SEQ ID NO:2, a GP/PT polypeptide of at least 395 amino acids that has at least 86% identity based on the Clustal method of alignment when compared to a glucose-6-phosphate/phosphate translocator polypeptide of SEQ ID NO:4, and a composition consisting of a GP/PTpolypeptide of at least 302 amino acids that has at least 93% identity based on the Clustal method of alignment when compared to a glucose-6-phosphate/phosphate translocator polypeptide of SEQ ID NO:6.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a GP/PT polypeptide in a host cell, preferably a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;
measuring the level a GP/PT polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of a GP/PT polypeptide in the host cell containing the isolated polynucleotide with the level of a GP/PT polypeptide in a host cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a GP/PT polypeptide gene, preferably a plant GP/PT polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, and 5 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a GP/PT amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a GP/PT polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a GP/PT, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a GP/PT, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of GP/PT in the transformed host cell; (c) optionally purifying the GP/PT expressed by the transformed host cell; (d) treating the GP/PT with a compound to be tested; and (e) comparing the activity of the GP/PT that has been treated with a test compound to the activity of an untreated GP/PT, thereby selecting compounds with potential for inhibitory activity.
The present invention relates to a composition comprising an isolated polynucleotide of the present invention.
The present invention relates to an isolated polynucleotide comprising at least one of 30 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5 and the complement of such sequences.
The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.
The present invention relates to a method for positive selection of a transformed cell comprising:
(a) transforming a plant cell with a chimeric gene of claim 5 or an expression cassette of the present invention; and
(b) growing the transformed plant cell, wherein the plant cell is a monocot or a dicot such as rice, soybean or wheat, under conditions allowing expression of the polynucleotide in an amount sufficient to complement a glucose-6-phosphate/phosphate translocator auxotroph to provide a positive selection means.